1. Field of the Invention
The present invention relates to the manufacture of enantiomerically-enriched preparations of amino acids and materials useful therefor. In particular, the present invention relates to an efficient industrial method to prepare natural or unnatural amino acids in high yield and high enantiomeric purity by stereoinversion of the opposite enantiomer using sequential reactions of biocatalytic oxidation and chemocatalytic reductive amination.
2. Description of the Background of the Invention
Amino acids typically exist in two mirror-image forms, known as “optical isomers” or “enantiomers”. This phenomenon is due to the asymmetric nature of these molecules, which underlies their molecular configurations referred to as left- and right-handed (chiral) forms. These forms are conventionally designated dextro (D) and levo (L) which display the optical property of shifting polarized light to either the right or the left as the light passes through such molecules. Both nature and industry use amino acids of one or the other chiral form, as appropriate, for given applications; hence, users of amino acids experience increased efficiency of use to the extent that the amino acids employed are optically-pure or -enriched for D- or L-amino acids, as appropriate. In industry, amino acid preparations are used for production of pharmaceutical, agrochemical, and fine chemical products.
To meet the industrial demand for these compounds, many methods have been described and developed to prepare amino acids with a high degree of optical purity. These methods include the physical separation or resolution of enantiomeric pairs using chromatographic or crystallization methods, biocatalytic resolution of enantiomers using enzymes, asymmetric synthesis of single enantiomers using chemical and/or biological catalysts, fermentation methods using engineered microbes, or selective stereoinversion methods. This last method combines a stereoselective enzyme (a biocatalyst) that forms an imine or keto acid intermediate and a non-selective metal catalyst (a chemocatalyst) that facilitates reduction of the intermediate species into a racemic amino acid mixture. Sequentially repeating this process results in a high degree of stereo-specific purity.
While each of the aforementioned approaches has been successful in specific instances, each has been limited by inherently low efficiency and/or narrow applicability to the broad family of amino acids required by industry. For example, fermentation methods are limited to the production of natural amino acids, whereas most of the amino acids required for pharmaceutical and agrochemical applications are unnatural. Resolution methods are limited in almost all cases to a maximum product yield of 50%, thereby incurring costs and generating waste in the forms of solvents and unreacted by-products at a minimum. Asymmetric synthesis of amino acids using chemical and biological catalysts is limited by many factors, including the narrow substrate ranges of the chemo- and biocatalysts employed in the process, limited access to starting materials, and greater expenses related to such starting materials.
Selective stereoinversion methods as currently practiced combine the biocatalyst and the chemocatalyst in a “one-pot” reaction. Accordingly, both the biocatalyzed oxidation and the chemocatalyzed reduction reactions operate under the same conditions. In practice, this limits the (potentially more robust) reduction reaction because it must be carried out under milder conditions amenable to the more sensitive biocatalytic reaction, such as a relatively moderate pH and a relatively low temperature. As a consequence, the activity of the metal catalyst is low, necessitating high catalyst usage to achieve reasonable rates of conversion. Neither does the oxidase enzyme operate optimally under current methodologies due to the compromise to use higher than optimal temperature conditions, for example, in order to lessen the detrimental impact on the metal catalyst activity under the biocatalyst-driven milder conditions.
Further drawbacks of the selective stereoinversion method as currently practiced includes the inhibitory effects of the biocatalyst and chemocatalyst on each other. The biocatalyst binds efficiently to the metal catalyst, whereupon both catalysts deactivate. Yet another drawback relates to safety in operating the selective stereoinversion method as a “one-pot” process. Oxygen gas is a requirement for the biocatalytic oxidation reaction. The following reduction reaction uses hydrogen gas as a co-substrate or produces some hydrogen gas as a byproducts of transfer hydrogenation. Therefore, both oxygen gas and hydrogen gas are present simultaneously in the same reaction vessel. At large scale, the possibility of a catastrophic explosion is high.
The current disclosure relates to a significant and surprising improvement of the selective stereoinversion method. This improved method, as disclosed below, overcomes the limitations of the previously described process by (i) avoiding the compromise conditions for the oxidation and reduction reactions, (ii) preventing the inhibition of both catalysts, and (iii) eliminating the requirement for simultaneous oxidizing and reducing conditions in the same reaction vessel. The process described herein also achieves a superior product yield. Additionally, as both reactions may be run under optimal conditions, the volumetric productivity (space-time yield) is significantly improved. Thus, the present invention greatly enhances the efficiency, scope, and safety of this process.